Efficient synthesis and anti-tubercular activity of a series of spirocycles: An exercise in open science

Article abstract of DOI:10.1371/journal.pone.0111782

Tuberculosis afflicts an estimated 2 billion people worldwide and causes 1.3 million deaths annually. Chemotherapeutic solutions rely on drugs developed many years ago, with only one new therapeutic having been approved in the last 40 years. Given the rise of drug-resistant strains, there is an urgent need for the development of a more robust drug development pipeline. GlaxoSmithKline recently placed the structures and activities of 177 novel anti-tubercular leads in the public domain, as well as the results of ongoing optimisation of some of the series. Since many of the compounds arose from screening campaigns, their provenance was unclear and synthetic routes were in many cases not reported. Here we present the efficient synthesis of several novel analogues of one family of the GSK compounds termed "Spiros"-using an oxa-Pictet-Spengler reaction. The new compounds are attractive from a medicinal chemistry standpoint and some were potent against the virulent strain, suggesting this class is worthy of further study. The research was carried out using open source methodology, providing the community with full access to all raw experimental data in real time.

Full text of DOI:10.1371/journal.pone.0111782

RESEARCH ARTICLE
Efficient Synthesis and Anti-Tubercular
Activity of a Series of Spirocycles: An
Exercise in Open Science
Katrina A. Badiola1, Diana H. Quan2, James A. Triccas2, Matthew H. Todd1*
1. School of Chemistry, The University of Sydney, New South Wales, Australia, 2. Microbial Pathogenesis and
Immunity Group, Department of Infectious Diseases and Immunology, The University of Sydney, New South
Wales, Australia
*matthew.todd@sydney.edu.au
OPEN ACCESS
Citation: Badiola KA, Quan DH, Triccas JA, Todd
MH (2014) Efficient Synthesis and Anti-Tubercular
Activity of a Series of Spirocycles: An Exercise in
Open Science. PLoS ONE 9(12): e111782. doi:10.
1371/journal.pone.0111782
Abstract
Tuberculosis afflicts an estimated 2 billion people worldwide and causes 1.3 million
deaths annually. Chemotherapeutic solutions rely on drugs developed many years
ago, with only one new therapeutic having been approved in the last 40 years.
Given the rise of drug-resistant strains, there is an urgent need for the development
of a more robust drug development pipeline. GlaxoSmithKline recently placed the
structures and activities of 177 novel anti-tubercular leads in the public domain, as
well as the results of ongoing optimisation of some of the series. Since many of the
compounds arose from screening campaigns, their provenance was unclear and
synthetic routes were in many cases not reported. Here we present the efficient
synthesis of several novel analogues of one family of the GSK compounds—
termed ‘‘Spiros’’—using an oxa-Pictet–Spengler reaction. The new compounds are
attractive from a medicinal chemistry standpoint and some were potent against the
virulent strain, suggesting this class is worthy of further study. The research was
carried out using open source methodology, providing the community with full
access to all raw experimental data in real time.
Editor: Kamyar Afarinkia, Univ of Bradford, United
Kingdom
Received: May 14, 2014
Accepted: September 30, 2014
Published: December 10, 2014
Copyright: ß 2014 Badiola et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. General synthetic and analytical meth-
ods are detailed in Text S1. Raw NMR data for all
compounds are available from The University of
Sydney eScholarship Repository [http://hdl.handle.
net/2123/10506]. The open electronic laboratory
notebook for experiments carried out April–August
2013 is available from The University of Sydney
eScholarship Repository [http://hdl.handle.net/
2123/10461]; all other experiments are sum-
marised in Spreadsheet S1.
Funding: This work was supported by the
University of Sydney (KAB, MHT) and the National
Health and Medical Research Council Centre of
Research Excellence on Tuberculosis Control
(APP1043225)(JAT). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Introduction
Infection by M. tuberculosis resulting in symptomatic tuberculosis (TB) can be
fatal without treatment. In 2012, TB was responsible for the deaths of 1.3 million
people and a further 8.6 million people were infected [1]. Globally, an estimated
two billion people carry latent TB and are susceptible to developing active TB.
Current first-line treatments include the ‘‘short-course-chemotherapy’’ regime,
which involves combinations of rifampicin, isoniazid, pyrazinamide and
Competing Interests: Matthew Todd is a PLOS
ONE Editorial Board Member (as an Associate
Editor). This does not alter the authors’ adherence
to PLOS ONE Editorial policies and criteria.
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Anti-TB Spirocycles from Open Science
ethambutol, taken over at least 6 months [2]. These drugs have been in use since
the 1960s; the recent FDA approval of bedaquiline [3] makes this drug the first
new treatment for TB to be approved in 40 years. The spread of partially- and
totally drug resistant strains makes the development of new treatments (preferably
targeting new cellular mechanisms) a priority [1].
GlaxoSmithKline (GSK) recently published the structures and anti-TB activities
of 177 small molecules as part of a deposition of open data [4]. These leads were
identified out of a pool of ,20000 molecules, chosen from the GSK corporate
compound collection based on favourable cell permeability and drug-like
parameters. Of the 177 leads, seven compounds contained a thiophene spirocycle
core; these were termed Spiros by GSK, represented by GSK2200150A (Figure 1).
The members of the Spiros series are excellent starting points for the
development of new anti-TB agents. The compounds were identified following a
number of screens that evaluated their inhibition of the growth of mycobacteria,
cytotoxicity and physical properties. The Spiros appear to affect an essential
membrane transport protein (MmpL3) of M. tuberculosis [5]. There are no
currently-approved drugs that target MmpL3, but four structurally dissimilar
compounds (C, Figure 1) have been identified as acting on MmpL3 [6–9] as well
as a more recent set of indoleamides [10]. In 2012 SQ109 completed a phase IIa
clinical trial for pulmonary TB [11]. The Spiros analogues are not overtly similar
in structure to these compounds; further investigation into the specific mode of
action at MmpL3 is clearly required, but it is a desirable property of any new
antitubercular compound that it should have a target different to existing
therapeutics.
Screening campaigns frequently use commercial libraries that understandably
lack synthetic provenance. The synthesis of the secondary amine spirocycle core
has been incompletely reported in the patent literature and a synthesis of the GSK
hit compound (GSK2200150A) was described in the academic literature but with
incomplete data and limited information on analog synthesis. [4–5, 12]. We
rationalised that Spiros analogues could be rapidly produced by first constructing
the core using an oxa-Pictet–Spengler reaction followed by final stage
diversification from the secondary amine 3 (Figure 2). This would allow the rapid
synthesis of new Spiros analogues in three steps and the faster progression of this
series in a hit-to-lead campaign.
We herein describe this work which was conducted using an electronic
laboratory notebook on the internet [13] and an open source research philosophy
that had shown efficiency gains in the discovery of a synthetic route to a drug used
in the treatment of schistosomiasis [14]. The licence governing such work is that
the research may be used for any purpose, including for financial gain, provided
the project is cited. The relevant laboratory notebook, containing a browseable
snapshot of the experiments and all the data for the period April–August 2013, has
been deposited online [15]. Data for remaining experiments (period late August–
December 2013) are included in Spreadsheet S1 rather than the electronic
laboratory notebook due to a local technical difficulty at the time these data were
collected.
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Figure 1. The GSK HTS campaign identified GSK2200150A, which is representative of the GSK Spiros family of anti-TB leads (A). (B) The optimised Spiros
analogue developed by GSK [5]. (C) Existing anti-tubercular candidates that have a mode of action that involves MmpL3.
doi:10.1371/journal.pone.0111782.g001
Results and Discussion
Synthesis of the Spiros Analogues
Our first synthetic approach was the use of compound 5 to make 3 with an oxa-
Pictet Spengler reaction (Figure 3). We adapted the patent procedure (A,
Figure 2. Potential for the rapid synthesis of Spiros analogues via a common 2 amine intermediate 3.
˚
(A) An existing oxa-Pictet–Spengler reaction can be used to form the spirocycle core (blue) as the 2 amine 3
˚
[12]. (B) Strategy to diversify from the 2 core 3 to produce Spiros analogues (C) with variation at the
˚
piperidine nitrogen (red).
doi:10.1371/journal.pone.0111782.g002
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Figure 3. Synthesis of the core 70 was attempted using conditions adapted from the literature [12]. (A)
N -Debenzylation of 4 was achieved under palladium-catalysed transfer hydrogenation conditions [17]. (B)
Reaction of the ketone 5 with thiopheneethanol 1 in the presence of strong Brønsted acids did not produce the
expected spirocycle 3.
doi:10.1371/journal.pone.0111782.g003
Figure 2) [12] by using 4-piperidone 5, instead of the corresponding ketal 2, since
substitution of the ketal for the ketone in the oxa-Pictet–Spengler reaction should
have a limited effect the reaction outcome [16]. The ketone is commercially
available but was easily prepared from commercial 4-benzylpiperidinone 4 (A,
Figure 3) [17] a compound that was itself later used. However, the harsh
conditions of the cyclization (excess triflic acid) resulted in the formation of
intractable mixtures.
Several reaction variables were explored to promote cyclisation, such as
reducing the amount of acid, using the less acidic methanesulfonic acid [18–19],
introducing a non-polar solvent (toluene or dioxane) and heating the reaction.
These efforts resulted in either partial recovery of the thiophene starting material
along with a complex product mixture or decomposition and the formation of
polar compounds that were not identified.
The ketal 2 may therefore be crucial in this transformation where precise tuning
of reactivity and conditions is necessary to promote conversion but not
decomposition. However, formation of this ketal would unfavourably introduce
another step in the synthesis (A, red, Figure 4). We therefore devised an alternate
strategy for accessing the secondary amine 3: carrying out the oxa-Pictet–Spengler
reaction using 4 prior to removal of the N-benzyl group (B).
The first step of the revised strategy proved effective; the N-benzylated core (6)
was obtained in consistently good yield over a number of repeats (A, Figure 5).
The reaction was promoted by 1.5 equivalents of methanesulfonic acid instead of
triflic acid, the former being easier to handle. To counter the reduced acidity, the
reaction temperature was increased. Lowering the acid loading or reaction time
gave a mixture of the product 6 and starting material; the ketone 4 was inseparable
from the product 6 in the post-reaction workup or by flash chromatography. The
methanesulfonic acid-mediated reaction initially carried out was the most
effective at cleanly obtaining the desired spirocycle 6.
The next step was the removal of the N-benzyl group from 6 to give the
secondary amine 3 (B to D, Figure 5). The N-benzylated spirocycle 6 was stable to
a variety of common palladium-catalysed hydrogenolysis conditions (B). The
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Anti-TB Spirocycles from Open Science
S
1
O
OXA-PS
REACTION
N
H
5
2
4
3
6
4
Figure 4. Re-evaluating the route to the 2 amine core 3 (blue). (A) The attempted route based on the patent literature procedure [12]. The dotted lines
˚
represent the additional step (red) required to attempt cyclisation strictly under the patent conditions. (B) The revised strategy: cyclise to give 6 followed by
debenzylation to give the 2 amine 3 (blue).
˚
doi:10.1371/journal.pone.0111782.g004
transfer hydrogenation conditions used in the preparation of 4-piperidinone 5
were ineffective; starting material 6 was recovered. Subsequent attempts were
made using different combinations of pressure (hydrogen gas up to 8.3 bar),
transfer hydrogenation and extended reaction times. In all attempts, starting
material was recovered with minimal loss of material.
We turned instead to debenzylation conditions mediated by 1-chloroethyl
chloroformate 7 (green), which gave the expected secondary amine 3 (blue) in
excellent yield (C, Figure 5). The debenzylation proceeds presumably via a
carbamate intermediate following the reaction of the starting material 6 with the
chloroformate 7 and loss of benzyl chloride [20–21]. Subsequent decarboxylation,
Figure 5. Executing the revised strategy towards the synthesis of spirocycle core as the 2 amine 3. (A) The acid-mediated cyclisation. (B) Attempts
˚
to synthesise the 2 amine using catalytic hydrogenolysis. (C) 1-chloroethyl chloroformate was effective at producing the secondary amine 3. (D) 2-
chloroethyl chloroformate resulted in incomplete deprotection of 6.
˚
doi:10.1371/journal.pone.0111782.g005
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promoted by the excess of methanol and reflux conditions, produced the desired
secondary amine 3 [20–21]. Isolation of the carbamate 9 when 2-chloroethyl
chloroformate 8 was used is consistent with the proposed mechanism (D); the
initial N-debenzylation step would be unaffected given the similar reactivity of the
chloroformate functional groups, but methanol attack on the secondary carbon to
lose the b-chloride would be less likely than attack on the tertiary carbon to lose
the a-chloride. The secondary amine 3 was obtained, ready for final stage
diversification in ,84% yield over two steps.
Diversification from the secondary amine 3 using reductive amination and
acylating conditions enabled the rapid synthesis of a variety of compounds in
moderate to good yield (Figure 6). The sodium triacetoxyborohydride-mediated
reductive amination procedure was adapted from the literature [22–23]. Acylation
of the amine 3 was achieved using aroyl chlorides. All candidate compounds were
designed to exhibit acceptable calculated logP values.
The acylation products 16 to 18 and the arylpyrrole 14 exhibited convoluted
NMR spectra. The 13C{1H}-NMR spectra of 16 to 18 contained the signals
expected from the carbon atoms in the carbonyl groups (165 to 175 ppm in
1
CDCl3) indicating formation of the amide bond, but H–13C{1H} Heteronuclear
Single Quantum Correlation (HSQC) spectroscopy was required to elucidate the
1
piperidine 13C{1H} region (A, Figure 7); the broad H signals correlated to the
aliphatic region of the 13C{1H} spectrum (B) were consistent with the piperidine
ring protons and all environments were accounted for. The broad signals observed
for the methylene protons on the oxygen-containing ring in the compounds
containing an exocyclic amide bond (i.e. compounds 16–18) most likely arise
from the chirality exhibited by the individual rotamers (C and D), which, though
in dynamic equilibrium via an achiral intermediate (B), make the protons
attached to these carbons diastereotopic. Additional experiments were carried out
on the acylated product 16, which showed temperature and magnetic field
dependence (E) consistent with rapid rotation of the amide bond; at higher fields
or lower temperatures peaks for the individual rotamers, and their more
convoluted splitting patterns, became clear.
Activity of the Spiros Analogues
The activities of the compounds containing the spirocycle core (3, 6, 9–18) were
determined against the virulent M.tuberculosis strain (H37Rv) (Table 1). Initially,
M. tuberculosis H37Rv was exposed to a single compound dose of 100 mM for 7
days, and survival was determined in comparison to vehicle-treated bacterial cells
using a Resazurin microtiter assay of growth inhibition [24]. The potency of
compounds displaying activity at 100 mM was determined by calculating the
concentration of drug inhibiting 50% of bacterial growth (IC50). Excellent
inhibitory activity against H37Rv for compounds containing the N-benzyl/
pyrrole-type centres (6, 10, 11, 13 and 14) was observed other than for 12; the
cyclohexyl-containing spirocycle 1, the secondary amine 3 and the N-acyl-type
spirocycles (9, 16–18) were inactive. The presence of the N-CH2-Ar group
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Anti-TB Spirocycles from Open Science
Figure 6. Diversification in the final step. The conditions for reductive amination (A) and acylation (B) of 3 to produce the final Spiros analogs 10–18 the
yields reported are of pure material isolated following flash chromatography on silica.
doi:10.1371/journal.pone.0111782.g006
appeared to be a necessary but not sufficient condition for significant activity
against H37Rv, but amide-type functionality was detrimental to the potency of the
molecules. Novel compounds 13 and 14 were the most potent with activity
comparable to the Spiros anti-TB compounds identified and developed by GSK
[4–5].
Toxicity of compounds displaying anti-H37Rv activity was assessed using the
human monocytic cell line THP1 (Table 1). The IC50 against THP was calculated
and was compared to the IC50 calculated against M. tuberculosis H37Rv.
Compounds 6, 10, 11 and 13 displayed THP1 toxicity at relatively high
concentrations (.50 mM), suggesting potential for the future development of
these compounds. Compounds 6 and 11 were less toxic than the original GSK
structure (10) yet were also less potent against H37Rv. Compound 14 was highly
active against H37Rv however was toxic against THP1 in the low mM range.
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Figure 7. The piperidine nitrogen signals of the acylated products can be visualised by HSQC
experiments. The 1H-NMR and 13C{1H}-NMR spectra respectively displayed on the x- and y-axes of the
HSQC spectra are projections of the corresponding one-dimensional NMR experiments and are displayed for
clarity. (A) The aliphatic region of the 1H–13C{1H} HSQC spectrum of 16; the red arrows indicate the 1H signals
corresponding to the piperidine protons (attached to the red ring of the structure). The corresponding
piperidine carbon signals on the y-axis (circled red) are unclear in the 13C{1H} spectrum. (B) Rotation around
the amide bond generates chiral rotamers (C and D) dictated by the p-system of the benzamide, which makes
the methylene protons in the oxygen-containing ring diastereotopic, (E) Variable magnetic field temperature
and NMR experiments showing coalescence of methylene signals for compound 16. At lower temperature the
diastereotopic oxygen-containing ring protons (E) exhibited higher order coupling, arising from the individual
rotamers (Figure S39) and the 13C{1H} NMR spectrum exhibited complete resolution of the piperidine ring
carbon signals (Figure S40). Raw data may be found in Dataset S1.
doi:10.1371/journal.pone.0111782.g007
Conclusion
A three-step synthesis of new TB drug leads is reported that will provide rapid
access to potent compounds that may be used in a future assessment of this series.
This should both aid in the synthesis of existing analogues for examination of
their pharmacokinetic properties and the synthesis of diverse new analogues in
this series to optimise potency and drug likeness as well as to further mechanism
of action studies. Future participants in such efforts are encouraged to adopt the
open platform that has already been developed for full data sharing and
collaboration.
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Table 1. Anti-tubercular activity of the synthesised Spiros analogues.
Entry
Compound
CLogP
0.83
3.1
H37Rv IC50 (mM)
.100
5
THP IC5 (mM)
1
3
n/a
200
n/a
100
.200
n/a
50
2
6
3
9
2.1
.100
1.25
4
10
11
12
13
14
15
16
17
18
3.0
5
3.0
10
6
2.2
.100
1.25
7
3.8
8
5.4*
3.2
1.25
6.3
n/a
n/a
n/a
n/a
9
.100
.100
.100
.100
10
11
12
1.7
2.6
2.6
Compounds with a minimum inhibitory concentration .25000 nM were not tested in the inhibition assay of starting concentration 2 mM. The CLogP were
calculated using ChemBioDraw Ultra 12.0.3 and the values are a guide only. CLogP values of rifampicin were also calculated using the same method for
consistency.
*The CLogP value was calculated for the free compound.
doi:10.1371/journal.pone.0111782.t001
Materials and Methods
General Chemistry
General synthetic and analytical methods are detailed in Text S1. Raw NMR data
for all compounds are available from The University of Sydney eScholarship
Repository [25]. The open electronic laboratory notebook for experiments carried
out April–August 2013 is available from The University of Sydney eScholarship
Repository [15]; all other experiments are summarised in Spreadsheet S1.
69,79-Dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 3
This compound exists in the literature but no data are reported [22]. To a stirred
solution of 6 (2.3 g, 7.7 mmol, 1 equiv.) in anhydrous THF (200 mL) under
argon at 278 C was added 1-chloroethyl chloroformate (1.6 mL, 15 mmol,
˚
2 equiv.). The reaction mixture was stirred for 30 min then allowed to warm to rt.
THF was removed under reduced pressure leaving a residual volume of ,10 mL
which was diluted with methanol (200 mL) and heated at reflux for 20 min. The
now clear brown solution was concentrated under reduced pressure to give the
crude product as a brown foam (2.8 g). The crude material was purified by flash
chromatography on silica (5:100:0.5, CH3OH:CH2Cl2:NH4OH, v/v to 10:100:1,
CH3OH:CH2Cl2:NH4OH, v/v) to give the title compound as a pale brown solid
(1.6 g, 99%); mp 86–88 C; nmax (film)/cm21 2953, 2923, 1072, 1050, 741, 654; dH
˚
(500 MHz; CDCl3) 7.06 (d, J55.2 Hz, 1 H), 6.79 (d, J55.2 Hz, 1 H), 3.93 (tapp
,
J55.4 Hz, 2 H), 3.06–3.01 (m, 2 H), 2.90–2.87 (m, 2 H), 2.82 (tapp, J55.3 Hz, 2
H), 1.85–1.78 (m, 4 H), 1.66 (brs, 1 H) (Figure S1); dC (126 MHz; CDCl3) 141.6,
1
132.5, 124.5, 122.4, 73.9, 59.1, 42.2, 37.0, 26.0 (Figure S2); H –13C HSQC
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Anti-TB Spirocycles from Open Science
1
(Figure S3) and H –13C{1H} HMBC (Figure S4) spectra also supplied; Anal.
Calcd. for C11H15NOS: C, 63.12; H, 7.22; N, 6.69. Found: C, 63.46; H, 7.56; N,
6.61.
1-Benzyl-69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 6
To a vigorously stirred solution of thiopheneethanol (0.50 mL, 4.5 mmol,
1 equiv.) and 1-benzyl-4-piperidinone (0.80 mL, 4.5 mmol, 1 equiv.) in toluene
(50 mL) was added methanesulfonic acid (0.29 mL, 4.5 mmol, 1 equiv.). The
reaction mixture was heated at reflux (oil bath at 130 C) for 16 h and allowed to
˚
cool to rt. The mixture was diluted with ethyl acetate (50 mL) and washed with
saturated NaHCO3 (30 mL). The aqueous fraction was extracted with ethyl
acetate (2630 mL), further basified with solid sodium hydroxide (4.0 g) and
extracted with ethyl acetate (2630 mL). The combined organic fractions were
dried (Na2SO4) and concentrated under reduced pressure to give the crude
product as a dark brown oil (1.7 g). The crude material was purified by flash
chromatography on silica (10–50% ethyl acetate/hexanes) to give the title
compound as a pale yellow oil (1.2 g, 86%); nmax (film)/cm21 2936, 2813, 1075,
854, 672; dH (500 MHz; CDCl3) 7.37–7.31 (m, 4 H), 7.27–7.24 (m, 1 H), 7.06 (d,
J55.2 Hz, 1 H), 6.82 (d, J55.2 Hz, 1 H), 3.93 (tapp, J55.4 Hz, 2 H), 3.56 (s, 2 H),
2.82 (tapp, J55.4 Hz, 2 H), 2.74–2.71 (m, 2 H), 2.43–2.38 (m, 2 H), 2.01–1.95 (m,
2 H), 1.86–1.82 (m, 2 H) (Figure S5); dC (126 MHz; CDCl3) 141.3, 138.6, 132.7,
1
129.4, 128.3, 127.1, 124.5, 122.3, 73.3, 63.5, 59.1, 49.2, 36.2, 26.0 (Figure S6); H
1
–13C HSQC (Figure S7) and H –13C{1H} HMBC (Figure S8) spectra also
supplied; HRMS (ESI) 300.14161 ([M+H]+), calcd. for C18H22NOS+ 300.14166.
Reductive amination: general procedure 1
This procedure was adapted from the literature [20] [22]. Compounds 10–15
were prepared using this method. To a stirred solution of secondary amine 3
(1 equiv.) and the appropriate aldehyde or ketone (1.1 equiv.) in anhydrous
dichloromethane (to 50 mM of 70) was added sodium triacetoxyborohydride
(1.5 equiv.). The mixture was stirred at rt under argon for 18–24 h then quenched
by pouring over saturated NaHCO3 solution. The aqueous phase was extracted
with dichloromethane (2 times). The combined organic fractions were dried
(Na2SO4) and concentrated under reduced pressure. The crude product was
purified by flash chromatography on silica to give the corresponding tertiary
amine product.
Acylation: general procedure 2
Compounds 16–18 were prepared using this method. A stirred solution of
secondary amine 3 (1 equiv.) and triethylamine (2 equiv.) in anhydrous
dichloromethane (to 0.2 M of 70) under argon was cooled in a brine ice bath. The
appropriate acid chloride (1 equiv.)—either benzoyl chloride, 2-bromobenzoyl
chloride or 4-bromobenzoyl chloride—was slowly added. The mixture was stirred
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Anti-TB Spirocycles from Open Science
at temperature for 15 min, slowly allowed to warm to rt and stirred for a further
for 12–15 h. The reaction was quenched by pouring over saturated NaHCO3
solution. The aqueous phase was extracted with dichloromethane (3 times). The
combined organic fractions were dried (Na2SO4) and concentrated under reduced
pressure. The crude product was purified by flash chromatography on silica to
give the corresponding amide product.
2-Chloroethyl 69,79-dihydrospiro[piperidine-4,49-thieno
[3,2-c]pyran]-1- carboxylate 9
This procedure was adapted from the literature [22]. To a stirred solution of 3
(0.40 g, 1.4 mmol, 1 equiv.) in anhydrous THF (35 mL) under argon at 278 C
˚
was added 2-chloroethyl chloroformate (0.18 mL, 1.7 mmol, 1.3 equiv.). The
reaction mixture was stirred at 278 C for 30 min then allowed to warm to rt. The
˚
solvent was removed under reduced pressure and the brownish residue was
suspended in methanol (40 mL) and heated at reflux for 1 h. The now clear
brown solution was concentrated under reduced pressure to give the crude
product as a yellow oil (,0.5 g). The crude material was purified by flash
chromatography on silica (10% ethyl acetate/CH2Cl2) to give the title compound as
a colourless oil that solidified when taken to 4 C (0.35 g, 82%); mp 57–60 C; n
˚
˚
max
(film)/cm21 2953, 2923, 1072, 1050, 741, 654; dH (500 MHz; CDCl3) 7.08 (d,
J55.2 Hz, 1 H), 6.72 (d, J55.2 Hz, 1 H), 4.36 (tapp, J55.7 Hz, 2 H), 4.04 (brs, 2
H), 3.94 (tapp, J55.4 Hz, 2 H), 3.71 (t, J55.6 Hz, 1 H), 3.21 (s, 1 H), 2.83 (t,
J55.4 Hz, 1 H), 1.84–1.81 (m, 1 H) (Figure S9); dC (126 MHz; CDCl3) 154.9,
140.3, 132.9, 124.1, 122.7, 73.2, 65.0, 59.4, 42.5, 39.9, 25.9 (Figure S10); HRMS
1
(ESI) 280.10010 ([M2Cl]+) calcd. for C14H18NO3S+ 280.10019; H –13C HSQC
1
(Figure S11) and H –13C{1H} HMBC (Figure S12) spectra also supplied; HRMS
(ESI) 338.05867 ([M+Na]+), calcd. for C14H18ClNO3SNa+ 338.05881; Anal. Calcd.
for C14H18ClNO3S: C, 53.24; H, 5.74; N, 4.44. Found: C, 53.21; H, 5.77; N, 4.40.
13C{1H}-NMR signals are missing or obscured due to rotamers around the
carbamate.
1-((2,3-Dihydrobenzo[b][1, 4]dioxin-6-yl)methyl)-69,
79-dihydrospiro[piperidine- 4,49-thieno[3,2-c]pyran] 10
Prepared according to general procedure 1. Following purification by flash
chromatography (1:100:0.1, CH3OH:CHCl3:NH4OH), the title compound was
obtained as a colourless oil that solidified to a colourless solid when taken to -
20 C (52 mg, 58%); mp 106–109 C; n
(film)/cm21 2926, 2813, 1505, 1068,
˚
886, 648; dH (500 MHz; CDCl3) 7.06 (d, J55.1 Hz, 1 H), 6.88 (sapp, 1 H), 6.84–
˚
max
6.79 (m, 3 H), 4.25 (s, 4 H), 3.92 (tapp, J55.3 Hz, 2 H), 3.45 (s, 2 H), 2.82 (tapp
,
J55.3 Hz, 2 H), 2.71 (brdapp, J511.2 Hz, 2 H), 2.37 (tapp, J511.9 Hz, 2 H), 1.97
(tdapp, J513.2, 4.0 Hz, 2 H), 1.84 (d, J513.0 Hz, 2 H) (Figure S13); dC (126 MHz;
CDCl3) 143.4, 142.7, 141.4, 132.7, 132.0, 124.6, 122.40, 122.30, 118.1, 117.0, 73.4,
64.51, 64.49, 62.9, 59.1, 49.1, 36.3, 26.0 (Figure S14); 1H –13C HSQC (Figure S15)
PLOS ONE | DOI:10.1371/journal.pone.0111782 December 10, 2014
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Anti-TB Spirocycles from Open Science
1
and H –13C{1H} HMBC (Figure S16) spectra also supplied; HRMS (ESI)
358.14712 ([M+H]+), calcd. for C20H24NO3S+ 358.14714; Anal. Calcd. for
C20H23NO3S: C, 67.20; H, 6.49; N, 3.92. Found: C, 66.55; H, 6.54; N, 3.88.
Discrepancy in CHN analysis noted, but data consistent with 4M+ H2O, i.e. calcd.
for C80H94N4O13S4 66.36; H, 6.54; N, 3.87.
1-(Benzo[d] [1, 3]dioxin-5-ylmethyl)-69,79-dihydrospiro[piperidine-
4,49-thieno [3,2-c]pyran] 11
Prepared according to general procedure 1. Following purification by flash
chromatography (2:100:0.2, CH3OH:CH2Cl2:NH4OH), the title compound was
obtained as a colourless oil that solidified to a colourless solid when taken to 4 C
˚
(37 mg, 44%); mp 81–84 C; nmax (film)/cm21 2923, 2813, 1501, 1487, 1440, 1242,
1074, 1036, 934, 853, 648; dH (500 MHz; CDCl3) 7.06 (d, J55.2 Hz, 1 H), 6.89
˚
(dapp, J51.0 Hz, 1 H), 6.81 (d, J55.2 Hz, 1 H), 6.78 (dd, J58.0, 1.0 Hz, 1 H), 6.75
(dapp, J58.0 Hz, 1 H), 3.92 (tapp, J55.4 Hz, 2 H), 3.47 (s, 2 H), 2.82 (tapp,
J55.4 Hz, 2 H), 2.72–2.70 (m, 2 H), 2.40–2.35 (m, 2 H), 2.00–1.94 (m, 2 H),
1.86–1.83 (m, 2 H) (Figure S17); dC (126 MHz; CDCl3) 147.7, 146.7, 141.3,
132.73, 132.56, 124.5, 122.48, 122.33, 109.8, 108.0, 101.0, 73.4, 63.3, 59.1, 49.1,
36.3, 26.0 (Figure S18); HRMS (ESI) 344.13146 ([M+H]+), calcd. for
C19H22NO3S+ 344.13149; Anal. Calcd. for C19H21NO3S: C, 66.45; H, 6.16; N, 4.08.
Found: C, 66.59; H, 6.26; N, 3.98.
4-((69,79-Dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl)
(2-methoxyphenol) 12
Prepared according to general procedure 1. Following purification by flash
chromatography (2:100:0.1, CH3OH:CH2Cl2:NH4OH), the title compound was
obtained as a colourless solid (41 mg, 44%); mp 170–172 C; n
(film)/cm21
˚
max
2927, 1517, 1277, 1265, 1071, 800, 732; dH (500 MHz; CDCl3) 7.04 (d, J55.2 Hz,
1 H), 6.90 (sapp, 1 H), 6.83–6.77 (m, 2+1 H), 3.92 (tapp, J55.4 Hz, 2 H), 3.83 (s, 3
H), 3.50 (s, 2 H), 2.82 (tapp, J55.4 Hz, 2 H), 2.76–2.74 (m, 2 H), 2.41–2.36 (m, 2
H), 2.83–1.83 (m, 2 H), 2.02–1.96 (m, 2 H) (Figure S19); dC (126 MHz; CDCl3)
146.7, 144.9, 141.1, 132.6, 129.9, 124.4, 122.38, 122.19, 114.2, 112.0, 73.3, 63.3,
1
59.0, 55.8, 49.0, 42.0, 35.9, 27.0, 25.9, 25.0 (Figure S20); H –13C HSQC (Figure
S21) spectrum also supplied; HRMS (ESI) 346.14711 ([M+H]+), calcd. for
C19H24NO3S+ 346.14714; Anal. Calcd. for C19H23NO3S: C, 66.06; H, 6.71; N, 4.05.
Found: C, 65.88; H, 6.91; N, 3.87.
1-(4-Chlorobenzyl)-69,79-dihydrospiro[piperidine-4,49-thieno
[3,2-c]pyran] 13
Prepared according to general procedure 1. Following purification by flash
chromatography (3:100:0.3, CH3OH:CHCl3:NH4OH), the title compound was
obtained as a colourless oil that solidified when taken to 4 C (79 mg, 64%); mp
˚
106–109 C; n
˚
(film)/cm21 2923, 2816, 1489, 1076, 1016, 854, 646; dH
max
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Anti-TB Spirocycles from Open Science
(500 MHz; CDCl3) 7.29 (sapp, 4 H), 7.07 (d, J55.2 Hz, 1 H), 6.81 (d, J55.2 Hz, 1
H), 3.92 (tapp, J55.4 Hz, 2 H), 3.52 (s, 2 H), 2.82 (tapp, J55.4 Hz, 2 H), 2.70–2.67
(m, 2 H), 2.42–2.36 (m, 2 H), 1.99–1.93 (m, 2 H), 1.86–1.83 (m, 2 H) (Figure
S22); dC (126 MHz; CDCl3) 141.2, 137.3, 132.8, 130.6, 128.5, 124.5, 122.4, 59.1,
49.2, 36.3, 26.0 (Figure S23); HRMS (ESI) 334.10268 ([M+H]+), calcd. for
C18H21ClNOS+ 334.10269; Anal. Calcd. for C18H20ClNOS: C, 64.75; H, 6.04; N,
4.20. Found: C, 64.56; H, 6.08; N, 4.16.
1-((1-(4-Fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-69,
79- dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] hydrochloride
14
Prepared according to general procedure 1. Following purification by flash
chromatography (2:100:0.2, CH3OH:CHCl3:NH4OH), the title compound was
obtained as a brown solid (23 mg, 22%); nmax (film)/cm21 2922, 1511, 1224,
1075, 852; dH (500 MHz; CDCl3) 7.18–7.14 (m, 3 H), 6.88 (s, 1 H), 6.02 (s, 1 H),
3.93 (tapp, J55.4 Hz, 2 H), 3.57 (brs, 2 H), 2.97 (brs, 1 H), 2.83 (tapp, J55.3 Hz, 2
H), 2.54 (s, 2 H), 2.23 (s, 1 H), 1.99 (s, 4 H), 1.91–1.88 (m, 3 H), 1.68 (s, 3 H)
(Figure S24); dC (126 MHz; CDCl3) 163.0, 161.0, 140.6, 135.0, 135.0, 132.7, 130.1,
130.1, 124.7, 122.6, 116.3, 116.1, 108.6, 72.9, 59.4, 54.4, 48.5, 35.3, 29.8, 26.0, 12.9,
11.1 (Figure S25); dF (471 MHz; CDCl3) -113.8; HRMS (ESI) 411.19012
([M2Cl]+), calcd. for C24H28FN2OS 411.19009; Anal. Calcd. for C24H28ClFN2OS:
C, 64.49; H, 6.31; N, 6.27. Found: C, 64.05; H, 6.14; N, 5.95.
1-Cyclohexyl-69,79-dihydrospiro[piperidine-4,49-thieno
[3,2-c]pyran] 15
Prepared according to general procedure 1. Following purification by flash
chromatography (0.8:100:0.08, CH3OH:CHCl3:NH4OH) the title compound was
obtained as a colourless solid (67 mg, 84%); mp 170–172 C; n
(film)/cm21
˚
max
2925, 2854, 1076, 647; dH (500 MHz; CDCl3) 7.05 (d, J55.2 Hz, 1 H), 6.84 (d,
J55.2 Hz, 1 H), 3.92 (t, J55.4 Hz, 2 H), 2.82 (t, J55.4 Hz, 2 H), 2.78–2.76 (m, 2
H), 2.65 (t, J511.6 Hz, 2 H), 2.38–2.34 (m, 1 H), 2.04–1.81 (m, 8 H), 1.66–1.62
(m, 1 H), 1.32–1.21 (m, 4 H), 1.15–1.08 (m, 1 H) (Figure S26); dC (126 MHz;
CDCl3) 141.2, 132.7, 124.7, 122.3, 73.6, 64.3, 59.1, 44.8, 36.5, 29.0, 26.5, 26.2, 26.0
(Figure S27); HRMS (ESI) 292.17301 ([M+H]+), calcd. for C17H26NOS+
292.17296; Anal. Calcd. for C17H25NOS: C, 70.06; H, 8.65; N, 4.81. Found: C,
69.51; H, 8.67; N, 4.70. Discrepancy in CHN analysis noted, but data consistent
with 5M + H2O i.e. calcd. for C85H127N5O6S5: C, 69.20; H, 8.68; N, 4.75.
69,79-Dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl)
methanone 16
Prepared according to general procedure 2. Following purification by flash
chromatography (1:100:0.1, CH3OH:CHCl3:NH4OH), the title compound was
obtained as a colourless solid (60 mg, 66%); mp 170–172 C; n
˚
(film)/cm21
max
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Anti-TB Spirocycles from Open Science
2951, 1432, 1050, 709; dH (500 MHz; CDCl3) 7.45–7.40 (m, 5 H), 7.10 (d,
J55.2 Hz, 1 H), 6.76 (d, J55.2 Hz, 1 H), 4.67–4.64 (m, 1 H), 3.97–3.94 (m, 2 H),
3.65–3.60 (m, 1 H), 3.50–3.45 (m, 1 H), 3.22–3.16 (m, 1 H), 2.85 (t, J55.4 Hz, 2
H), 2.01–1.75 (m, 4 H) (Figure S28); dC (126 MHz; CDCl3; 300 K) 170.5, 140.1,
136.4, 133.0, 129.7, 128.6, 127.1, 124.1, 122.9, 73.5, 59.5, 25.9 (Figure S29); dC
(126 MHz; CDCl3; 270 K) 170.5, 139.9, 136.0, 133.0, 129.7, 128.6, 127.0, 124.1,
122.9, 73.4, 59.5, 43.7, 38.1, 36.7, 35.5, 25.8; HRMS (ESI) 336.10287 ([M+Na]+),
calcd. for C18H19NO2SNa+ 336.10287; Anal. Calcd. for C18H19NO2S: C, 68.98; H,
6.11; N, 4.47. Found: C, 68.03; H, 6.05; N, 4.44. Discrepancy in CHN analysis
noted, but data consistent with 4M + H2O i.e. calcd. for C72H78N4O9S4: C, 68.00;
H, 6.18; N, 4.41. 13C{1H} signals are missing or obscured due to rotamers around
1
the amide bond. The signals were visualised using H –13C HSQC spectroscopy
(Figure S30) and completely resolved at 270 K (Figure S40). Variable temperature
NMR data for this compound may be found in Dataset S1.
(4-Bromophenyl)(69,79-dihydrospiro[piperidine-4,49-thieno
[3,2-c]pyran]-1-yl) methanone 17
Prepared according to general procedure 2. Following purification by flash
chromatography (1:100:0.1, CH3OH:CHCl3:NH4OH), the title compound was
obtained as a colourless solid (60 mg, 66%); mp 170–172 C; n
(film)/cm21
˚
max
2923, 1628, 1433, 1072; dH (500 MHz; CDCl3) 7.56–7.52 (m, 2 H), 7.39–7.30 (m,
2 H), 7.10 (d, J55.2 Hz, 1 H), 6.74 (d, J55.2 Hz, 1 H), 4.65–4.58 (m, 1 H), 3.95–
3.92 (m, 2 H), 3.62–3.44 (m, 2 H), 3.23–3.15 (m, 1 H), 2.85 (tapp, J55.4 Hz, 2 H),
2.02–1.83 (m, 2 H), 1.83–1.72 (m, 2 H) (Figure S31); dC (126 MHz; CDCl3) 169.5,
139.9, 135.1, 133.1, 131.8, 128.8, 124.00, 123.97, 122.9, 73.4, 59.5, 25.9 (Figure
1
1
S32); H –13C HSQC (Figure S33) and H –13C{1H} HMBC (Figure S34) spectra
also supplied; HRMS (ESI) 416.01134 ([M+Na]+), calcd. for C18H1881BrNOSNa+
416.01134; Anal. Calcd. for C18H18BrNO2S: C, 55.11; H, 4.62; N, 3.57. Found: C,
55.88; H, 4.89; N, 3.45. 13C{1H} signals are missing or obscured due to rotamers
around the amide bond. Discrepancy in CHN analysis noted; data match sample
containing ,10% des-brominated compound, but this was not observed in the
1H NMR spectrum for this sample.
(2-Bromophenyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-
c]pyran]-1-yl) methanone 18
Prepared according to general procedure 2. Following purification by flash
chromatography (1:100:0.1, CH3OH:CH2Cl2:NH4OH), the title compound was
obtained as a colourless solid (81 mg, 73%); mp 48–52 C; nmax (film)/cm21 2932,
˚
1634, 1436, 1073, 768; dH (500 MHz; CDCl3) 7.60–7.54 (m, 1 H), 7.38–7.31 (m, 2
H), 7.24 (dd, J513.3, 6.7 Hz, 2 H), 7.08 (dd, J54.8, 2.5 Hz, 1 H), 6.72 (d,
J55.2 Hz, 1 H), 4.73–4.66 (m, 1 H), 3.93 (tq, J510.0, 4.9 Hz, 2 H), 3.55 (td,
J512.9, 3.2 Hz,), 3.42–3.34 (m, 1 H), 3.28–3.12 (m, 2 H), 2.83 (t, J54.9 Hz, 2 H),
2.10 (td, J513.5, 5.0 Hz, 1 H), 1.99 (dd, J59.8, 3.6 Hz, 1 H), 1.94–1.90 (m, 1 H),
PLOS ONE | DOI:10.1371/journal.pone.0111782 December 10, 2014
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Anti-TB Spirocycles from Open Science
1.81–1.69 (m, 2 H), 1.26 (t, J57.2 Hz, 1 H), 1.05 (t, J57.1 Hz, ,1 H) (Figure
S35); dC (126 MHz; CDCl3) 167.8, 167.6, 140.0, 139.9, 138.9, 138.5, 138.4, 133.4,
132.9, 132.8, 132.8, 130.3, 130.2, 130.0, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5,
124.1, 123.9, 122.8, 122.8, 119.4, 119.2, 73.4, 73.3, 59.5, 43.5, 42.8, 42.5, 39.0, 37.7,
1
37.5, 36.6, 36.3, 35.6, 35.5, 31.0, 25.8, 14.0, 12.6 (Figure S36); H –13C HSQC
1
(Figure S37) and H –13C{1H} HMBC (Figure S38) spectra also supplied; HRMS
(ESI) 414.01338 ([M+Na]+), calcd. for C18H18BrNO2SNa+ 414.01338; Anal. Calcd.
for C18H18BrNO2S: C, 55.11; H, 4.62; N, 3.57. Found: C, 55.36; H, 4.36; N, 3.59.
1
The additional 13C{1H} and H signals are due to rotamers around the amide
bond.
Resazurin assay of growth inhibition
Compounds were tested for activity at either single concentration (100 mM) or
serially diluted in 10 mL of purified H2O in triplicate in 96 well microtiter plates.
M. tuberculosis H37Rv was grown in complete Middlebrook 7H9 media (Bacto,
Australia) containing albumin, dextrose and catalase (ADC), 20% Tween 80 and
50% glycerol (Sigma-Aldrich, Australia). A bacterial suspension (90 mL) at
OD600nm of 0.001 was added to the wells and incubated for 7 days. Resazurin
(10 mL; 0.05%(w/v); Sigma-Aldrich, Australia) was then added, incubated for
24 h at 37 C, and fluorescence measured at 590 nm using a FLUOstar Omega
˚
microplate reader (BMG Labtech, Germany). After subtraction of background
fluorescence from all wells, the percentage mycobacterial survival was determined
by comparing the fluorescence of wells containing compounds compared to
control wells not treated with compound.
Compound intracellular efficacy and toxicity
THP1 cells (TIB-202R), a human monocyte cell line (American Type Culture
Collection, USA), were grown in complete Dulbecco’s Modified Eagle Media
(DMEM; LifeTechnologies, Australia) including 10% fetal bovine serum (FBS),
200 mM L-glutamine (LifeTechnologies, Australia), and 1 mM HEPES buffer
solution (LifeTechnologies, Australia). Cells (16105) in media containing 50 ng/
mL phorbol 12-myristate 13-acetate (PMA) were added to 96-well plates and were
then incubated for 48 h at 37 C to allow adherence and differentiation.
˚
Compounds (1.56–200 mM) were added to the wells and incubated for 4 days at
37 C. Then 0.05%(w/v) resazurin (4 h) was added and the fluorescence measured.
˚
Cell viability was calculated as percentage fluorescence in comparison to untreated
cells.
Supporting Information
1
Figure S1. H NMR (500 MHz, CDCl3) spectrum of 69,79-dihydrospiro[piper-
idine-4,49-thieno[3,2-c]pyran] 3.
doi:10.1371/journal.pone.0111782.s001 (PDF)
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Anti-TB Spirocycles from Open Science
Figure S2. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 69,79-dihydrospir-
o[piperidine-4,49-thieno[3,2-c]pyran] 3.
doi:10.1371/journal.pone.0111782.s002 (EPS)
1
Figure S3. H –13C HSQC (500 MHz, CDCl3) spectrum of 69,79-dihydrospir-
o[piperidine-4,49-thieno[3,2-c]pyran] 3.
doi:10.1371/journal.pone.0111782.s003 (EPS)
1
Figure S4. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of 69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 3.
doi:10.1371/journal.pone.0111782.s004 (EPS)
1
Figure S5. H NMR (500 MHz, CDCl3) spectrum of 1-benzyl-69,79-dihydros-
piro[piperidine-4,49-thieno[3,2-c]pyran] 6.
doi:10.1371/journal.pone.0111782.s005 (PDF)
Figure S6. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-benzyl-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 6.
doi:10.1371/journal.pone.0111782.s006 (EPS)
1
Figure S7. H –13C HSQC (500 MHz, CDCl3) spectrum of 1-benzyl-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 6.
doi:10.1371/journal.pone.0111782.s007 (EPS)
1
Figure S8. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of 1-benzyl-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 6.
doi:10.1371/journal.pone.0111782.s008 (EPS)
1
Figure S9. H NMR (500 MHz, CDCl3) spectrum of 2-chloroethyl 69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-carboxylate 9.
doi:10.1371/journal.pone.0111782.s009 (EPS)
Figure S10. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 2-chloroethyl 69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-carboxylate 9.
doi:10.1371/journal.pone.0111782.s010 (EPS)
1
Figure S11. H –13C HSQC (500 MHz, CDCl3) spectrum of 2-chloroethyl 69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-carboxylate 9.
doi:10.1371/journal.pone.0111782.s011 (EPS)
1
Figure S12. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of 2-chloroethyl
69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-carboxylate 9.
doi:10.1371/journal.pone.0111782.s012 (EPS)
1
Figure S13. H NMR (500 MHz, CDCl3) spectrum of 1-((2,3-dihydroben-
zo[b][1, 4]dioxin-6-yl)methyl)-69,79-dihydrospiro[piperidine- 4,49-thieno[3,2-
c]pyran] 10.
doi:10.1371/journal.pone.0111782.s013 (EPS)
Figure S14. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-((2,3-dihydroben-
zo[b][1, 4]dioxin-6-yl)methyl)-69,79-dihydrospiro[piperidine- 4,49-thieno[3,2-
c]pyran] 10.
doi:10.1371/journal.pone.0111782.s014 (EPS)
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Anti-TB Spirocycles from Open Science
1
Figure S15. H –13C HSQC (500 MHz, CDCl3) spectrum of 1-((2,3-dihydro-
benzo[b][1, 4]dioxin-6-yl)methyl)-69,79-dihydrospiro[piperidine- 4,49-
thieno[3,2-c]pyran] 10.
doi:10.1371/journal.pone.0111782.s015 (EPS)
1
Figure S16. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of 1-((2,3-
dihydrobenzo[b][1, 4]dioxin-6-yl)methyl)-69,79-dihydrospiro[piperidine- 4,49-
thieno[3,2-c]pyran] 10.
doi:10.1371/journal.pone.0111782.s016 (EPS)
Figure S17. 1H NMR (500 MHz, CDCl3) spectrum of 1-(benzo[d][1, 3]dioxin-5-
ylmethyl)-69,79-dihydrospiro[piperidine- 4,49-thieno [3,2-c]pyran] 11.
doi:10.1371/journal.pone.0111782.s017 (EPS)
Figure S18. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-(benzo[d][1, 3]
dioxin-5-ylmethyl)-69,79-dihydrospiro[piperidine- 4,49-thieno [3,2-c]pyran] 11.
doi:10.1371/journal.pone.0111782.s018 (EPS)
1
Figure S19. H NMR (500 MHz, CDCl3) spectrum of 4-((69,79-dihydrospir-
o[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.
doi:10.1371/journal.pone.0111782.s019 (EPS)
Figure S20. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 4-((69,79-dihydros-
piro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.
doi:10.1371/journal.pone.0111782.s020 (EPS)
1
Figure S21. H –13C HSQC (500 MHz, CDCl3) spectrum of 4-((69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.
doi:10.1371/journal.pone.0111782.s021 (EPS)
1
Figure S22. H NMR (500 MHz, CDCl3) spectrum of 1-(4-chlorobenzyl)-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 13.
doi:10.1371/journal.pone.0111782.s022 (EPS)
Figure S23. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-(4-chlorobenzyl)-
69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 13.
doi:10.1371/journal.pone.0111782.s023 (EPS)
Figure S24. 1H NMR (500 MHz, CDCl3) spectrum of 1-((1-(4-fluorophenyl)-2,5-
dimethyl-1H-pyrrol-3-yl)methyl)-69,79- dihydrospiro[piperidine-4,49-thieno[3,2-
c]pyran] hydrochloride 14.
doi:10.1371/journal.pone.0111782.s024 (EPS)
Figure S25. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-((1-(4-fluorophe-
nyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-69,79- dihydrospiro[piperidine-4,49-
thieno[3,2-c]pyran] hydrochloride 14.
doi:10.1371/journal.pone.0111782.s025 (PDF)
1
Figure S26. H NMR (500 MHz, CDCl3) spectrum of 1-cyclohexyl-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 15.
doi:10.1371/journal.pone.0111782.s026 (EPS)
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Anti-TB Spirocycles from Open Science
Figure S27. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 1-cyclohexyl-69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran] 15.
doi:10.1371/journal.pone.0111782.s027 (EPS)
1
Figure S28. H NMR (500 MHz, CDCl3) spectrum of 69,79-dihydrospiro[piper-
idine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.
doi:10.1371/journal.pone.0111782.s028 (EPS)
Figure S29. 13C{1H} NMR (126 MHz, CDCl3) spectrum of 69,79-dihydrospir-
o[piperidine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.
doi:10.1371/journal.pone.0111782.s029 (EPS)
1
Figure S30. H –13C HSQC (500 MHz, CDCl3) spectrum of 69,79-dihydrospir-
o[piperidine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.
doi:10.1371/journal.pone.0111782.s030 (EPS)
1
Figure S31. H NMR (500 MHz, CDCl3) spectrum of (4-bromophenyl)(69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 17.
doi:10.1371/journal.pone.0111782.s031 (EPS)
Figure S32. 13C{1H} NMR (126 MHz, CDCl3) spectrum of (4-bromophe-
nyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 17.
doi:10.1371/journal.pone.0111782.s032 (EPS)
1
Figure S33. H –13C HSQC (500 MHz, CDCl3) spectrum of (4-bromophe-
nyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 17.
doi:10.1371/journal.pone.0111782.s033 (EPS)
1
Figure S34. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of (4-bromo-
phenyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone
17.
doi:10.1371/journal.pone.0111782.s034 (EPS)
1
Figure S35. H NMR (500 MHz, CDCl3) spectrum of (2-bromophenyl)(69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 18.
doi:10.1371/journal.pone.0111782.s035 (EPS)
Figure S36. 13C{1H} NMR (126 MHz, CDCl3) spectrum of (2-bromophe-
nyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 18.
doi:10.1371/journal.pone.0111782.s036 (EPS)
1
Figure S37. H –13C HSQC (500 MHz, CDCl3) spectrum of (2-bromophe-
nyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone 18.
doi:10.1371/journal.pone.0111782.s037 (EPS)
1
Figure S38. H –13C{1H} HMBC (500 MHz, CDCl3) spectrum of (2-bromo-
phenyl)(69,79-dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl) methanone
18.
doi:10.1371/journal.pone.0111782.s038 (EPS)
1
Figure S39. H NMR (CDCl3) spectra (4.5–2.5 ppm) of 69,79-dihydrospiro[pi-
peridine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16 at variable tem-
perature and magnetic field strength.
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Anti-TB Spirocycles from Open Science
doi:10.1371/journal.pone.0111782.s039 (EPS)
Figure S40. Overlay of the 13C{1H} NMR (126 MHz, CDCl3) spectra of 69,79-
dihydrospiro[piperidine-4,49-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16 at
300 K and 270 K.
doi:10.1371/journal.pone.0111782.s040 (EPS)
Text S1. General synthetic and chemical analysis methods.
doi:10.1371/journal.pone.0111782.s041 (RTF)
Spreadsheet S1. Summary of the experimental the experimental data.
doi:10.1371/journal.pone.0111782.s042 (XLSX)
Dataset S1. Variable Temperature NMR Data for Compound 16.
doi:10.1371/journal.pone.0111782.s043 (ZIP)
Acknowledgments
Synthesis of the novel arylpyrrole 14 was possible through the kind provision of
the relevant aldehyde by Dr. Alice Williamson (The University of Sydney). We are
grateful to technical assistance from Dr. Ian Luck (NMR spectroscopy), Mr. John
Twyman (electronic lab notebook) and Dr. Nick Proschogo (mass spectrometry);
elemental analyses were performed at The Campbell Microanalytical Laboratory,
University of Otago. The electronic lab notebook used was the open source
Labtrove software developed at the University of Southampton (www.labtrove.
org); we thank the Labtrove team for help in creating the browseable snapshot.
Author Contributions
Conceived and designed the experiments: KAB JAT MHT. Performed the
experiments: KAB DHQ. Analyzed the data: KAB DHQ JAT MHT. Contributed
reagents/materials/analysis tools: KAB DHQ. Wrote the paper: KAB JAT MHT.
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